In this proposal we focus on exploring the folding of two proteins, the isozymes of aspartate aminotransferase, which are encoded by the nuclear genome and synthesized in cytoplasmic free polysomes. One of these proteins (cytosolic, cAAT) remains in the cytoplasmic compartment for its whole life span; the other (mitochondrial, mAAT) ends up residing in the mitochondrial matrix. Furthermore, mAAT is synthesized as a precursor protein (pmAAT) with an additional amino terminal extension known as the presequence or signal peptide. The molecular mechanisms for the intracellular targeting and translocation of proteins have been actively investigated, but very little is known regarding the early stages of sorting which are probably dictated by information encoded in the amino acid sequence. Mounting evidence indicates that molecular chaperones, a group of proteins ubiquitous in all types of cells, might have a role in the control of these events. Our main goal is to elucidate whether the folding of a protein in a given intracellular compartment depends on the information carried in its amino acid sequence, on the particular set of molecular chaperones present in that compartment or on both. The immediate aims are: 1) Characterize the role of a) the presequence peptide in pmAAT and b) of other defined segments in mature cAAT and mAAT in the folding process of these proteins as it occurs in buffer alone (in vitro) and in the presence of cytoplasmic extracts (in situ conditions). This analysis will make use of chimeric, engineered proteins prepared by fusion of the signal peptide to the cytosolic isozyme or by exchanging segments of a few pertinent regions between the two isozymes. 2) Analyze the interaction of the wild type and chimeric forms alluded to in aim 1 with specific chaperones (starting with Grovel and hsp70), to identify contact sites and folding states of the proteins in the complexes.